Infant feeding formulas comprising probiotic micro-organisms

ABSTRACT

The present invention relates to the field of infant nutrition. In particular, the present invention relates to infant feeding formulas comprising probiotic micro-organisms. These probiotic micro-organisms may be non-replicating probiotic micro-organisms such as bioactive heat treated probiotic micro-organisms, for example.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a National Stage of International ApplicationNo. PCT/EP2010/056419, filed on May 11, 2010, which claims priority toEuropean Patent Application No. 09159925.8, filed on May 11, 2009 andEuropean Patent Application No. 09159929.0, filed on May 11, 2009, theentire contents of which are being incorporated herein by reference.

The present invention relates to the field of infant nutrition. Inparticular, the present invention relates to infant feeding formulascomprising probiotic micro-organisms. These probiotic micro-organismsmay be non-replicating probiotic micro-organisms such as bioactive heattreated probiotic micro-organisms, for example.

Breast milk is the ideal food for healthy growth and development ofbabies. In 2001 the World Health Organization (WHO) changed itsrecommended duration of exclusive breastfeeding from 4 to 6 months to 6months, therefore breastfeeding should be encouraged and promotedaccordingly.

However, not all mothers are able to feed their babies on breast milkalone for 6 months, or, for various reasons, they choose not to.

The global rate of exclusive breastfeeding was 46% in 1999, according toUNICEF. However, this situation varies dramatically from country tocountry. e.g. the reported exclusive breast feeding rate in Liberia is73% while in Kenya reaches only 5%.

Many factors could have an impact on exclusive breastfeeding patternsand they are usually linked to economical, social and culturalcircumstances.

Also a significant part of all mothers is unable to provide sufficientamounts of milk to their babies, so that complementary nutrition isneeded.

Infant formula is the best nutritional alternative for those babies whoare not breastfed or not sufficiently breastfed.

The Federal Food, Drug, and Cosmetic Act (FFDCA) defines infant formulaas “a food which purports to be or is represented for special dietaryuse solely as a food for infants by reason of its simulation of humanmilk or its suitability as a complete or partial substitute for humanmilk” (FFDCA 201(z)).

One important function of early nutrition of infants is to generate ahealthy gut flora and to develop a strong immune system.

A healthy gut flora will contribute to a functional GI tract, which inturn will help to properly digest ingested food and will reduce stomachache in newborns.

It would hence be desirable to further improve the immune boostingeffect of infant formulas, to further improve its anti-inflammatoryeffect and to facilitate digestion.

Hence, there is a need in the art for an infant feeding formula thatallows to provide infants with nutrition that is as close as possible tomother's milk. Such an infant feeding formula should have an improvedimmune boosting effect, an anti-inflammatory effect and/or shouldfacilitate digestion. It would be preferred if this was achieved byusing natural ingredients that are safe to administer without sideeffects and that are easy to incorporate into infant feeding formulasusing state of the art industrial techniques.

The present inventors have addressed this need. It was hence theobjective of the present invention to improve the state of the art andto provide an infant feeding formula that satisfies the needs expressedabove.

The present inventors were surprised to see that they could achieve thisobject by the subject matter of the independent claim. The dependantclaims further develop the idea of the present invention.

Accordingly, the present inventors propose to provide an infant feedingformula comprising probiotics to be administered to infants.

FDA regulations define infants as persons not more than 12 months old(Title 21, Code of Federal Regulations 21 CFR 105.3(e)).

Probiotics were found to be able to provide their health benefits in theframework of infant feeding formulas Additionally, e.g. bifidobacteria,are present in breast milk and are part of what gives breast milk itsnaturally protective properties.

Hence, adding probiotic micro-organisms to infant-feeding formulas wouldallow them to more closely resemble breast milk.

However, as in particular powdered infant feeding formulas to bereconstituted with water usually have a shelf life that exceeds theshelf life of, e.g., yoghurt drinks comprising probiotics, probioticsare usually not added to such infant feeding formulas, because ofuncertainties that the viability of the probiotics can be ensured duringan extended shelf life.

The present inventors were now able to show that even non-replicatingprobiotics can provide the health benefits of probiotics and may evenhave improved benefits.

Hence one embodiment of the present invention is an infant feedingformula to be administered to an infant as only nutrition source or asonly complementary nutrition source in addition to breastfeeding thatprovides complete nutrition to the infant and comprises probioticmicro-organisms.

A composition provides complete nutrition if an infant obtains allrequired nutrients from this composition and no additional food sourcesare required.

The infant feeding formula may be provided as liquid composition readyto be administered or as dried composition to be reconstituted withwater prior to use.

If provided as a dried composition it is preferred that the compositionhas a water activity of below 0.2, preferably below 0.15 to furtherincrease shelf stability. Most bacteria, for example, do not grow atwater activities below 0.91, and most molds cease to grow at wateractivities below 0.80.

Water activitiy (a_(w)) is a measurement of the energy status of thewater in a system. It is defined as the vapour pressure of the waterdivided by that of pure water. Consequently, distilled water has a waterpressure of 1.

The infant feeding formula in accordance with the present invention mayhave a caloric density in the range of 62-68 kcal/100 ml.

Typically, an infant feeding formula may comprise a protein source in anamount of 1.5-2.8 g/100 kcal, a carbohydrate source in an amount of10-12 g/100 kcal, and a lipid source in an amount of 5-5.5 g/100 kcal.

The protein source may consist of whey proteins and casein. For examplea ratio of whey to casein in the range of about 70:30 may be used.However for increased protein needs whey and casein may be used in aratio in the range of 20:80 to 50:50.

The carbohydrate source may consist essentially of Lactose. However,also ratios of Lactose and Maltodextrin in the range of 3:1 to 1:1 maybe used, for example.

The infant feeding formula of the present invention may comprise 0.2-0.3g LC-PUFA/100 g fatty acids. The LC-PUFA may for example comprise acombination of ARA and DHA. Formulas containing DHA and ARA have beenshown to provide visual and mental development similar to that of thebreastfed infant.

The infant feeding formula of the present invention may also contain1.5-2.5 mg nucleotides per 100 mL formula. Nucleotides and their basesare not considered ‘essential’ because they can be synthesised by theinfant body from simpler compounds. At certain times, however, theprocesses of synthesis may not be able to meet demand, for example,during periods of rapid cell turnover as in normal growth or in gutdisease. At these times, the body relies more heavily on dietary sourcesof nucleotides.

The infant feeding formula may comprise in part or only non-replicatingprobiotic micro-organisms.

The inventors were surprised to see that, e.g., in terms of an immuneboosting effect and/or in terms of an anti-inflammatory effectnon-replicating probiotic microorganisms may even be more effective thanreplicating probiotic microorganisms.

This is surprising since probiotics are often defined as “livemicro-organisms that when administered in adequate amounts confer healthbenefits to the host” (FAO/WHO Guidelines). The vast majority ofpublished literature deals with live probiotics. In addition, severalstudies investigated the health benefits delivered by non-replicatingbacteria and most of them indicated that inactivation of probiotics,e.g. by heat treatment, leads to a loss of their purported healthbenefit (Rachmilewitz, D., et al., 2004, Gastroenterology 126:520-528;Castagliuolo, et al., 2005, FEMS Immunol. Med. Microbiol. 43:197-204;Gill, H. S. and K. J. Rutherfurd, 2001, Br. J. Nutr. 86:285-289; Kaila,M., et al., 1995, Arch. Dis. Child 72:51-53.). Some studies showed thatkilled probiotics may retain some health effects (Rachmilewitz, D., etal., 2004, Gastroenterology 126:520-528; Gill, H. S. and K. J.Rutherfurd, 2001, Br. J. Nutr. 86:285-289), but clearly, livingprobiotics were regarded in the art so far as more performing.

The infant feeding formula according to the present invention maycomprise probiotic micro-organisms in any effective amount, for examplein an amount corresponding to about 10⁶ to 10¹² cfu/g dry weight.

The probiotic micro-organisms may be non-replicating probioticmicro-organisms.

“Non-replicating” probiotic micro-organisms include probiotic bacteriawhich have been heat treated. This includes micro-organisms that areinactivated, dead, non-viable and/or present as fragments such as DNA,metabolites, cytoplasmic compounds, and/or cell wall materials.

“Non-replicating” means that no viable cells and/or colony forming unitscan be detected by classical plating methods. Such classical platingmethods are summarized in the microbiology book: James Monroe Jay,Martin J. Loessner, David A. Golden. 2005. Modern food microbiology. 7thedition, Springer Science, New York, N.Y. 790 p. Typically, the absenceof viable cells can be shown as follows: no visible colony on agarplates or no increasing turbidity in liquid growth medium afterinoculation with different concentrations of bacterial preparations(‘non replicating’ samples) and incubation under appropriate conditions(aerobic and/or anaerobic atmosphere for at least 24 h).

Probiotics are defined for the purpose of the present invention as“Microbial cell preparations or components of microbial cells with abeneficial effect on the health or well-being of the host.” (Salminen S,Ouwehand A. Benno Y. et al “Probiotics: how should they be defined”Trends Food Sci. Technol. 1999:10 107-10).

The possibility to use non-replicating probiotic micro-organisms offersseveral advantages. In severely immuno-compromised infants, the use oflive probiotics may be limited in exceptional cases due to a potentialrisk to develop bacteremia. Non-replicating probiotics may be usedwithout any problem.

Additionally, the provision of non-replicating probiotic micro-organismsallows the hot reconstitution while retaining health benefit.

The infant feeding formulas of the present invention comprise probioticmicro-organisms and/or non-replicating probiotic micro-organisms in anamount sufficient to at least partially produce a health benefit. Anamount adequate to accomplish this is defined as “a therapeuticallyeffective dose”. Amounts effective for this purpose will depend on anumber of factors known to those of skill in the art such as the weightand general health state of the infant, and on the effect of the foodmatrix.

In prophylactic applications, infant feeding formulas according to theinvention are administered to a consumer susceptible to or otherwise atrisk of a disorder in an amount that is sufficient to at least partiallyreduce the risk of developing that disorder. Such an amount is definedto be “a prophylactic effective dose”. Again, the precise amounts dependon a number of factors such as the infant's state of health and weight,and on the effect of the food matrix.

Those skilled in the art will be able to adjust the therapeuticallyeffective dose and/or the prophylactic effective dose appropriately.

In general the infant feeding formula of the present invention containsprobiotic micro-organisms and/or non-replicating probioticmicro-organisms in a therapeutically effective dose and/or in aprophylactic effective dose.

Typically, the therapeutically effective dose and/or the prophylacticeffective dose is in the range of about 0,005 mg-1000 mg probioticmicro-organisms and/or non-replicating, probiotic micro-organisms perdaily dose.

In terms of numerical amounts, the “short-time high temperature” treatednon-replicating micro-organisms may be present in the infant feedingformula in an amount corresponding to between 10⁴ and 10¹² equivalentcfu/g of the dry composition. Obviously, non-replicating micro-organismsdo not form colonies, consequently, this term is to be understood as theamount of non replicating micro-organisms that is obtained from 10⁴ and10¹² cfu/g replicating bacteria. This includes micro-organisms that areinactivated, non-viable or dead or present as fragments such as DNA orcell wall or cytoplasmic compounds. In other words, the quantity ofmicro-organisms which the infant feeding formula contains is expressedin terms of the colony forming ability (cfu) of that quantity ofmicro-organisms as if all the micro-organisms were alive irrespective ofwhether they are, in fact, non replicating, such as inactivated or dead,fragmented or a mixture of any or all of these states.

Preferably the non-replicating micro-organisms are present in an amountequivalent to between 10⁴ to 10⁹ cfu/g of dry infant feeding formula,even more preferably in an amount equivalent to between 10⁵ and 10⁹cfu/g of dry infant feeding formula.

The probiotics may be rendered non-replicating by any method that isknown in the art.

The technologies available today to render probiotic strainsnon-replicating are usually heat-treatment, γ-irradiation, UV light orthe use of chemical agents (formalin, paraformaldehyde).

It would be preferred to use a technique to render probioticsnon-replicating that is relatively easy to apply under industrialcircumstances in the food industry.

Most products on the market today that contain probiotics are heattreated during their production. It would hence be convenient, to beable to heat treat probiotics either together with the produced productor at least in a similar way, while the probiotics retain or improvetheir beneficial properties or even gain a new beneficial property forthe consumer.

However, inactivation of probiotic micro-organisms by heat treatments isassociated in the literature generally with an at least partial loss ofprobiotic activity.

The present inventors have now surprisingly found, that renderingprobiotic micro-organisms non-replicating, e.g., by heat treatment, doesnot result in the loss of probiotic health benefits, but—to thecontrary—may enhance existing health benefits and even generate newhealth benefits.

Hence, one embodiment of the present invention is an infant feedingformula wherein the non-replicating probiotic micro-organisms wererendered non-replicating by a heat-treatment.

Such a heat treatment may be carried out at least 71.5° C. for at least1 second.

Long-term heat treatments or short-term heat treatments may be used.

In industrial scales today usually short term heat treatments, such asUHT-like heat treatments are preferred. This kind of heat treatmentreduces bacterial loads, and reduces the processing time, therebyreducing the spoiling of nutrients.

The inventors demonstrate for the first time that probioticsmicro-organisms, heat treated at high temperatures for short timesexhibit anti-inflammatory immune profiles regardless of their initialproperties. In particular either a new anti-inflammatory profile isdeveloped or an existing anti-inflammatory profile is enhanced by thisheat treatment.

It is therefore now possible to generate non replicating probioticmicro-organisms with anti-inflammatory immune profiles by using specificheat treatment parameters that correspond to typical industriallyapplicable heat treatments, even if live counterparts are notanti-inflammatory strains.

Hence, for example, the heat treatment may be a high temperaturetreatment at about 71.5-150° C. for about 1-120 seconds. The hightemperature treatment may be a high temperature/short time (HTST)treatment or a ultra-high temperature (UHT) treatment.

The probiotic micro-organisms may be subjected to a high temperaturetreatment at about 71.5-150° C. for a short term of about 1-120 seconds.

More preferred the micro-organisms may be subjected to a hightemperature treatment at about 90-140° C., for example 90°-120° C., fora short term of about 1-30 seconds.

This high temperature treatment renders the micro-organisms at least inpart non-replicating.

The high temperature treatment may be carried out at normal atmosphericpressure but may be also carried out under high pressure. Typicalpressure ranges are form 1 to 50 bar, preferably from 1-10 bar, evenmore preferred from 2 to 5 bar. Obviously, it is preferred if theprobiotics are heat treated in a medium that is either liquid or solid,when the heat is applied. An ideal pressure to be applied will thereforedepend on the nature of the composition which the micro-organisms areprovided in and on the temperature used.

The high temperature treatment may be carried out in the temperaturerange of about 71.5-150° C., preferably of about 90-120° C., even morepreferred of about 120-140° C.

The high temperature treatment may be carried out for a short term ofabout 1-120 seconds, preferably, of about 1-30 seconds, even morepreferred for about 5-15 seconds.

This given time frame refers to the time the probiotic micro-organismsare subjected to the given temperature. Note, that depending on thenature and amount of the composition the micro-organisms are provided inand depending on the architecture of the heating apparatus used, thetime of heat application may differ.

Typically, however, the infant feeding formula of the present inventionand/or the micro-organisms are treated by a high temperature short time(HTST) treatment, flash pasteurization or a ultra high temperature (UHT)treatment.

A UHT treatment is Ultra-high temperature processing or a ultra-heattreatment (both abbreviated UHT) involving the at least partialsterilization of a composition by heating it for a short time, around1-10 seconds, at a temperature exceeding 135° C. (275° F.), which is thetemperature required to kill bacterial spores in milk. For example,processing milk in this way using temperatures exceeding 135° C. permitsa decrease of bacterial load in the necessary holding time (to 2-5 s)enabling a continuous flow operation.

There are two main types of UHT systems: the direct and indirectsystems. In the direct system, products are treated by steam injectionor steam infusion, whereas in the indirect system, products are heattreated using plate heat exchanger, tubular heat exchanger or scrapedsurface heat exchanger. Combinations of UHT systems may be applied atany step or at multiple steps in the process of product preparation.

A HTST treatment is defined as follows (High Temperature/Short Time):Pasteurization method designed to achieve a 5-log reduction, killing99.9999% of the number of viable micro-organisms in milk. This isconsidered adequate for destroying almost all yeasts, molds and commonspoilage bacteria and also ensure adequate destruction of commonpathogenic heat resistant organisms. In the HTST process milk is heatedto 71.7° C. (161° F.) for 15-20 seconds.

Flash pasteurization is a method of heat pasteurization of perishablebeverages like fruit and vegetable juices, beer and dairy products. Itis done prior to filling into containers in order to kill spoilagemicro-organisms, to make the products safer and extend their shelf life.The liquid moves in controlled continuous flow while subjected totemperatures of 71.5° C. (160° F.) to 74° C. (165° F.) for about 15 to30 seconds.

For the purpose of the present invention the term “short time hightemperature treatment” shall include high-temperature short time (HTST)treatments, UHT treatments, and flash pasteurization, for example.

Since such a heat treatment provides non-replicating probiotics with animproved anti-inflammatory profile, the infant feeding formula of thepresent invention may be for use in the prevention or treatment ofinflammatory disorders.

The inflammatory disorders that can be treated or prevented by theinfant feeding formula of the present invention are not particularlylimited. For example, they may be selected from the group consisting ofacute inflammations such as sepsis; burns; and chronic inflammation,such as inflammatory bowel disease, e.g., Crohn's disease, ulcerativecolitis, pouchitis; necrotizing enterocolitis; skin inflammation, suchas UV or chemical-induced skin inflammation, eczema, reactive skin;irritable bowel syndrome; eye inflammation; allergy, asthma; andcombinations thereof.

If long term heat treatments are used to render the probioticmicro-organisms non-replicating, such a heat treatment may be carriedout in the temperature range of about 70-150° C. for about 3 minutes-2hours, preferably in the range of 80-140° C. from 5 minutes-40 minutes.

While the prior art generally teaches that bacteria renderednon-replicating by long-term heat-treatments are usually less efficientthan live cells in terms of exerting their probiotic properties, thepresent inventors were able to demonstrate that heat-treated probioticsare superior in stimulating the immune system compared to their livecounterparts.

The present invention relates also to an infant feeding formulacomprising probiotic micro-organisms that were rendered non-replicatingby a heat treatment at at least about 70° C. for at least about 3minutes.

The immune boosting effects of non-replicating probiotics were confirmedby in vitro immunoprofiling. The in vitro model used uses cytokineprofiling from human Peripheral Blood Mononuclear Cells (PBMCs) and iswell accepted in the art as standard model for tests of immunomodulatingcompounds (Schultz et al., 2003, Journal of Dairy Research 70, 165-173;Taylor et al., 2006, Clinical and Experimental Allergy, 36, 1227-1235;Kekkonen et al., 2008, World Journal of Gastroenterology, 14, 1192-1203)

The in vitro PBMC assay has been used by several authors/research teamsfor example to classify probiotics according to their immune profile,i.e. their anti- or pro-inflammatory characteristics (Kekkonen et al.,2008, World Journal of Gastroenterology, 14, 1192-1203). For example,this assay has been shown to allow prediction of an anti-inflammatoryeffect of probiotic candidates in mouse models of intestinal colitis(Foligne, B., et al., 2007, World J. Gastroenterol. 13:236-243).Moreover, this assay is regularly used as read-out in clinical trialsand was shown to lead to results coherent with the clinical outcomes(Schultz et al., 2003, Journal of Dairy Research 70, 165-173; Taylor etal., 2006, Clinical and Experimental Allergy, 36, 1227-1235).

Allergic diseases have steadily increased over the past decades and theyare currently considered as epidemics by WHO. In a general way, allergyis considered to result from an imbalance between the Th1 and Th2responses of the immune system leading to a strong bias towards theproduction of Th2 mediators. Therefore, allergy can be mitigated,down-regulated or prevented by restoring an appropriate balance betweenthe Th1 and Th2 arms of the immune system. This implies the necessity toreduce the Th2 responses or to enhance, at least transiently, the Th1responses. The latter would be characteristic of an immune boostresponse, often accompanied by for example higher levels of IFNγ, TNF-αand IL-12. (Kekkonen et al., 2008, World Journal of Gastroenterology,14, 1192-1203; Viljanen M. et al., 2005, Allergy, 60, 494-500)

The infant feeding formula of the present invention allows it hence totreat or prevent disorders that are related to a compromised immunedefence.

Consequently, the disorders linked to a compromised immune defence thatcan be treated or prevented by the infant feeding formula of the presentinvention are not particularly limited.

For example, they may be selected from the group consisting ofinfections, in particular bacterial, viral, fungal and/or parasiteinfections; phagocyte deficiencies; low to severe immunodepressionlevels such as those induced by stress or immunodepressive drugs,chemotherapy or radiotherapy; natural states of less immunocompetentimmune systems such as those of the neonates; allergies; andcombinations thereof.

The infant feeding formula described in the present invention allows italso to enhance a infant's response to vaccines, in particular to oralvaccines.

Any amount of non-replicating micro-organisms will be effective.However, it is generally preferred, if at least 90%, preferably, atleast 95%, more preferably at least 98%, most preferably at least 99%,ideally at least 99.9%, most ideally all of the probiotics arenon-replicating.

In one embodiment of the present invention all micro-organisms arenon-replicating.

Consequently, in the infant feeding formula of the present invention atleast 90%, preferably, at least 95%, more preferably at least 98%, mostpreferably at least 99%, ideally at least 99.9%, most ideally all of theprobiotics may be non-replicating.

All probiotic micro-organisms may be used for the purpose of the presentinvention.

For example, the probiotic micro-organisms may be selected from thegroup consisting of bifidobacteria, lactobacilli, propionibacteria, orcombinations thereof, for example Bifidobacteriurm longum,Bifidobacterium lactis, Bifidobacterium animalis, Bifidobacterium breve,Bifidobacterium infantis, Bifidobacterium adolescentis, Lactobacillusacidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillussalivarius, Lactobacillus reuteri, Lactobacillus rhamnosus,Lactobacillus johnsonii, Lactobacillus plantarum, Lactobacillusfermentum, Lactococcus lactis, Streptococcus thermophilus, Lactococcuslactis, Lactococcus diacetylactis, Lactococcus cremoris, Lactobacillusbulgaricus, Lactobacillus helveticus, Lactobacillus delbrueckii,Escherichia coli and/or mixtures thereof.

The composition in accordance with the present invention may, forexample comprise probiotic micro-organisms selected from the groupconsisting of Bifidobacterium longum NCC 3001, Bifidobacterium longumNCC 2705, Bifidobacterium breve NCC 2950, Bifidobacterium lactis NCC2818, Lactobacillus johnsonii La1, Lactobacillus paracasei NCC 2461,Lactobacillus rhamnosus NCC 4007, Lactobacillus reuteri DSM17938,Lactobacillus reuteri ATCC55730, Streptococcus thermophilus NCC 2019,Streptococcus thermophilus NCC 2059, Lactobacillus casei NCC 4006,Lactobacillus acidophilus NCC 3009, Lactobacillus casei ACA-DC 6002 (NCC1825), Escherichia coli Nissle, Lactobacillus bulgaricus NCC 15,Lactococcus lactis NCC 2287, or combinations thereof.

All these strains were either deposited under the Budapest treaty and/orare commercially available.

The strains have been deposited under the Budapest treaty as follows:

Bifidobacterium longum NCC 3001: ATCC BAA-999 (deposited on Jan. 29,2001 at the Institut Pasteur, 28 rue du Docteur Roux, F-75024 ParisCedex 15, France)

Bifidobacterium longum NCC 2705: CNCM I-2618

Bifidobacterium breve NCC 2950: CNCM I-3865 (deposited on Nov. 15, 2007at the Institut Pasteur, 28 rue du Docteur Roux, F-75024 Paris Cedex 15,France)

Bifidobacterium lactis NCC 2818: CNCM 1-3446 (deposited on Jun. 7, 2005at the Institut Pasteur, 28 rue du Docteur Roux, F-75024 Paris Cedex 15,France)

Lactobacillus paracasei NCC 2461: CNCM 1-2116 (deposited on Jan. 12,1999 at the Institut Pasteur, 28 rue du Docteur Roux, F-75024 ParisCedex 15, France)

Lactobacillus rhamnosus NCC 4007: CGMCC 1.3724 (deposited in October2004 at the China General Microbiological Culture Collection Center,Chinese Academy of Sciences, P.O. Box 2714, Beijing, China 100080)

Streptococcus themophilus NCC 2019: CNCM 1-1422 (deposited on May 18,1994 at the Institut Pasteur, 28 rue du Docteur Roux, F-75024 ParisCedex 15, France)

Streptococcus themophilus NCC 2059: CNCM 1-4153 (deposited on Apr. 24,2009 at the Institut Pasteur, 28 rue du Docteur Roux, F-75024 ParisCedex 15, France)

Lactococcus lactis NCC 2287: CNCM 1-4154 (deposited on Apr. 24, 2009 atthe Institut Pasteur, 28 rue du Docteur Roux, F-75024 Paris Cedex 15,France)

Lactobacillus casei NCC 4006: CNCM 1-1518 (deposited on Jun. 12, 2008 atthe Institut Pasteur, 28 rue du Docteur Roux, F-75024 Paris Cedex 15,France)

Lactobacillus casei NCC 1825: ACA-DC 6002

Lactobacillus acidophilus NCC 3009: ATCC 700396

Lactobacillus bulgaricus NCC 15: CNCM 1-1198 (deposited on Apr. 2, 1992at the Institut Pasteur, 28 rue du Docteur Roux, F-75024 Paris Cedex 15,France)

Lactobacillus johnsonii La1: CNCM 1-1225 (deposited on Jun. 30, 1992 atthe Institut Pasteur, 28 rue du Docteur Roux, F-75024 Paris Cedex 15,France)

Lactobacillus reuteri DSM17938: DSM17938

Lactobacillus reuteri ATCC55730: ATCC55730

Escherichia coli Nissle 1917: DSM 6601

Those skilled in the art will understand that they can freely combineall features of the present invention described herein, withoutdeparting from the scope of the invention as disclosed.

Further advantages and features of the present invention are apparentfrom the following Examples and Figures.

FIGS. 1A and B show the enhancement of the anti-inflammatory immuneprofiles of probiotics treated with “short-time high temperatures”.

FIG. 2 shows non anti-inflammatory probiotic strains that becomeanti-inflammatory, i.e. that exhibit pronounced anti-inflammatory immuneprofiles in vitro after being treated with “short-time hightemperatures”.

FIGS. 3A and B show probiotic strains in use in commercially availableproducts that exhibit enhanced or new anti-inflammatory immune profilesin vitro after being treated with “short-time high temperatures”.

FIGS. 4A and B show dairy starter strains (i.e. Lc1 starter strains)that exhibits enhanced or new anti-inflammatory immune profiles in vitroupon heat treatment at high temperatures.

FIG. 5 shows a non anti-inflammatory probiotic strain that exhibitsanti-inflammatory immune profiles in vitro after being treated with HISTtreatments.

FIG. 6: Principal Component Analysis on PBMC data (IL-12p40, IFN-γ,TNF-α, IL-10) generated with probiotic and dairy starter strains intheir live and heat treated (140° C. for 15 second) forms. Each dotrepresents one strain either live or heat treated identified by its NCCnumber or name.

FIG. 7 shows IL-12p40/IL-10 ratios of live and heat treated (85° C., 20min) strains. Overall, heat treatment at 85° C. for 20 min leads to anincrease of IL-12p40/IL-10 ratios as opposed to “short-time hightemperature” treatments of the present invention (FIGS. 1, 2, 3, 4 and5).

FIG. 8 shows the enhancement of in vitro cytokine secretion from humanPBMCs stimulated with heat treated bacteria.

FIG. 9 shows the percentage of diarrhea intensity observed inOVA-sensitized mice challenged with saline (negative control),OVA-sensitized mice challenged with OVA (positive control) andOVA-sensitized mice challenged with OVA and treated with heat-treated orlive Bifidobacterium breve NCC2950. Results are displayed as thepercentage of diarrhea intensity (Mean±SEM calculated from 4 independentexperiments) with 100% of diarrhea intensity corresponding to thesymptoms developed in the positive control (sensitized and challenged bythe allergen) group.

EXAMPLE 1 Methodology

Bacterial Preparations:

The health benefits delivered by live probiotics on the host immunesystem are generally considered to be strain specific. Probioticsinducing high levels of IL-10 and/or inducing low levels ofpro-inflammatory cytokines in vitro (PBMC assay) have been shown to bepotent anti-inflammatory strains in vivo (Foligné, B., et al., 2007,World J. Gastroenterol. 13:236-243).

Several probiotic strains were used to investigate the anti-inflammatoryproperties of heat treated probiotics. These were Bifidobacterium longumNCC 3001, Bifidobacterium longum NCC 2705, Bifidobacterium breve NCC2950, Bifidobacterium lactis NCC 2818, Lactobacillus paracasei NCC 2461,Lactobacillus rhamnosus NCC 4007, Lactobacillus casei NCC 4006,Lactobacillus acidophilus NCC 3009, Lactobacillus casei ACA-DC 6002 (NCC1825), and Escherichia coli Nissle. Several starter culture strainsincluding some strains commercially used to produce Nestlé Lc1 fermentedproducts were also tested: Streptococcus thermophilus NCC 2019,Streptococcus thermophilus NCC 2059, Lactobacillus bulgaricus NCC 15 andLactococcus lactis NCC 2287.

Bacterial cells were cultivated in conditions optimized for each strainin 5-15 L bioreactors. All typical bacterial growth media are usable.Such media are known to those skilled in the art. When pH was adjustedto 5.5, 30% base solution (either NaOH or Ca(OH)₂) was addedcontinuously. When adequate, anaerobic conditions were maintained bygassing headspace with CO₂ . E. coli was cultivated under standardaerobic conditions.

Bacterial cells were collected by centrifugation (5,000×g, 4° C.) andre-suspended in phosphate buffer saline (PBS) in adequate volumes inorder to reach a final concentration of around 10⁹-10¹⁰ cfu/ml. Part ofthe preparation was frozen at −80° C. with 15% glycerol. Another part ofthe cells was heat treated by:

-   -   Ultra High Temperature: 140° C. for 15 sec; by indirect steam        injection.    -   High Temperature Short Time (HTST): 74° C., 90° C. and 120° C.        for 15 sec by indirect steam injection    -   Long Time Low Temperature (85° C., 20 min) in water bath

Upon heat treatment, samples were kept frozen at −80° C. until use.

In Vitro Immunoprofiling of Bacterial Preparations:

The immune profiles of live and heat treated bacterial preparations(i.e. the capacity to induce secretion of specific cytokines from humanblood cells in vitro) were assessed. Human peripheral blood mononuclearcells (PBMCs) were isolated from blood filters. After separation by celldensity gradient, mononuclear cells were collected and washed twice withHank's balanced salt solution. Cells were then resuspended in Iscove'sModified Dulbecco's Medium (IMDM, Sigma) supplemented with 10% foetalcalf serum (Bioconcept, Paris, france), 1% L-glutamine (Sigma), 1%penicillin/streptomycin (Sigma) and 0.1% gentamycin (Sigma). PBMCs(7×10⁵ cells/well) were then incubated with live and heat treatedbacteria (equivalent 7×10⁶ cfu/well) in 48 well plates for 36 h. Theeffects of live and heat treated bacteria were tested on PBMCs from 8individual donors splitted into two separated experiments. After 36 hincubation, culture plates were frozen and kept at −20° C. untilcytokine measurement. Cytokine profiling was performed in parallel (i.e.in the same experiment on the same batch of PBMCs) for live bacteria andtheir heat-treated counterparts.

Levels of cytokines (IFN-γ, IL-12p40, TNF-α and IL-10) in cell culturesupernatants after 36 h incubation were determined by ELISA (R&D DuoSetHuman IL-10, BD OptEIA Human IL12p40, BD OptEIA Human TNFα, BD OptEIAHuman IFN-γ) following manufacturer's instructions. IFN-γ, IL-12p40 andTNF-α are pro-inflammatory cytokines, whereas IL-10 is a potentanti-inflammatory mediator. Results are expressed as means (pg/ml)+/−SEMof 4 individual donors and are representative of two individualexperiments performed with 4 donors each. The ratio IL-12p40/IL-10 iscalculated for each strain as a predictive value of in vivoanti-inflammatory effect (Foligné, B., et al., 2007, World J.Gastroenterol. 13:236-243).

Numerical cytokine values (pg/ml) determined by ELISA (see above) foreach strain were transferred into BioNumerics v5.10 software (AppliedMaths, Sint-Martens-Latem, Belgium). A Principal Component Analysis(PCA, dimensioning technique) was performed on this set of data.Subtraction of the averages over the characters and division by thevariances over the characters were included in this analysis.

Results

Anti-Inflammatory Profiles Generated by Ultra High Temperature(UHT)/High Temperature Short Time (HTST)-Like Treatments

The probiotic strains under investigation were submitted to a series ofheat treatments (Ultra High Temperature (UHT), High Temperature ShortTime (HTST) and 85° C. for 20 min) and their immune profiles werecompared to those of live cells in vitro. Live micro-organisms(probiotics and/or dairy starter cultures) induced different levels ofcytokine production when incubated with human PBMC (FIGS. 1, 2, 3, 4 and5). Heat treatment of these micro-organisms modified the levels ofcytokines produced by PBMC in a temperature dependent manner.“Short-time high temperature” treatments (120° C. or 140° C. for 15″)generated non replicating bacteria with anti-inflammatory immuneprofiles (FIGS. 1, 2, 3 and 4). Indeed, UHT-like treated strains (140°C., 15 sec) induced less pro-inflammatory cytokines (TNF-α, IFN-γ,IL-12p40) while maintaining or inducing additional IL-10 production(compared to live counterparts). The resulting IL-12p40/IL-10 ratioswere lower for any UHT-like treated strains compared to live cells(FIGS. 1, 2, 3 and 4). This observation was also valid for bacteriatreated by HTST-like treatments, i.e. submitted to 120° C. for 15 sec(FIGS. 1, 2, 3 and 4), or 74° C. and 90° C. for 15 sec (FIG. 5). Heattreatments (UHT-like or HTST-like treatments) had a similar effect on invitro immune profiles of probiotic strains (FIGS. 1, 2, 3 and 5) anddairy starter cultures (FIG. 4). Principal Component Analysis on PBMCdata generated with live and heat treated (140° C., 15″) probiotic anddairy starter strains revealed that live strains are spread all alongthe x axis, illustrating that strains exhibit very different immuneprofiles in vitro, from low (left side) to high (right side) inducers ofpro-inflammatory cytokines. Heat treated strains cluster on the leftside of the graph, showing that pro-inflammatory cytokines are much lessinduced by heat treated strains (FIG. 6). By contrast, bacteria heattreated at 85° C. for 20 min induced more pro-inflammatory cytokines andless IL-10 than live cells resulting in higher IL-12p40/IL-10 ratios(FIG. 7).

Anti-Inflammatory Profiles are Enhanced or Generated by UHT-Like andHTST-Like Treatments.

UHT and HTST treated strains exhibit anti-inflammatory profilesregardless of their respective initial immune profiles (live cells).Probiotic strains known to be anti-inflammatory in vivo and exhibitinganti-inflammatory profiles in vitro (B. longum NCC 3001, B. longum NCC2705, B. breve NCC 2950, B. lactis NCC 2818) were shown to exhibitenhanced anti-inflammatory profiles in vitro after “short-time hightemperature” treatments. As shown in FIG. 1, the IL-12p40/IL-10 ratiosof UHT-like treated Bifidobacterium strains were lower than those fromthe live counterparts, thus showing improved anti-inflammatory profilesof UHT-like treated samples. More strikingly, the generation ofanti-inflammatory profiles by UHT-like and HTST-like treatments was alsoconfirmed for non anti-inflammatory live strains. Both live L. rhamnosusNCC 4007 and L. paracasei NCC 2461 exhibit high IL-12p40/IL-10 ratios invitro (FIGS. 2 and 5). The two live strains were shown to be notprotective against TNBS-induced colitis in mice. The IL-12p40/IL-10ratios induced by L. rhamnosus NCC 4007 and L. paracasei NCC 2461 weredramatically reduced after “short-time high temperature” treatments (UHTor HTST) reaching levels as low as those obtained with Bifidobacteriumstrains. These low IL-12p40/IL-10 ratios are due to low levels ofIL-12p40 production combined with no change (L. rhamnosus NCC 4007) or adramatic induction of IL-10 secretion (L. paracasei NCC 2461) (FIG. 2).

As a consequence:

-   -   Anti-inflammatory profiles of live micro-organisms can be        enhanced by UHT-like and HTST-like heat treatments (for        instance B. longum NCC 2705, B. longum NCC 3001, B. breve NCC        2950, B. lactis NCC 2818)    -   Anti-inflammatory profiles can be generated from non        anti-inflammatory live micro-organisms (for example L. rhamnosus        NCC 4007, L. paracasei NCC 2461, dairy starters S. thermophilus        NCC 2019) by UHT-like and HTST-like heat treatments.    -   Anti-inflammatory profiles were also demonstrated for strains        isolated from commercially available products (FIGS. 3 A & B)        including a probiotic E. coli strain.

The impact of UHT/HTST-like treatments was similar for all testedprobiotics and dairy starters, for example lactobacilli, bifidobacteriaand streptococci.

UHT/HTST-like treatments were applied to several lactobacilli,bifidobacteria and streptococci exhibiting different in vitro immuneprofiles. All the strains induced less pro-inflammatory cytokines afterUHT/HTST-like treatments than their live counterparts (FIGS. 1, 2, 3, 4,5 and 6) demonstrating that the effect of UHT/HIST-like treatments onthe immune properties of the resulting non replicating bacteria can begeneralized to all probiotics, in particular to lactobacilli andbifidobacteria and specific E. coli strains and to all dairy startercultures in particular to streptococci, lactococci and lactobacilli.

EXAMPLE 2 Methodology

Bacterial Preparations:

Five probiotic strains were used to investigate the immune boostingproperties of non-replicating probiotics: 3 bifidobacteria (B. longumNCC3001, B. lactis NCC2818, B. breve NCC2950) and 2 lactobacilli (L.paracasei NCC2461, L. rhamnosus NCC4007).

Bacterial cells were grown on MRS in batch fermentation at 37° C. for16-18 h without pH control. Bacterial cells were spun down (5,000×g, 4°C.) and resuspended in phosphate buffer saline prior to be diluted insaline water in order to reach a final concentration of around 10E10cfu/ml. B. longum NCC3001, B. lactis NCC2818, L. paracasei NCC2461, L.rhamnosus NCC4007 were heat treated at 85° C. for 20 min in a waterbath. B. breve NCC2950 was heat treated at 90° C. for 30 minutes in awater bath. Heat treated bacterial suspensions were aliquoted and keptfrozen at −80° C. until use. Live bacteria were stored at −80° C. inPBS-glycerol 15% until use.

In Vitro Immunoprofiling of Bacterial Preparations

The immune profiles of live and heat treated bacterial preparations(i.e. the capacity to induce secretion of specific cytokines from humanblood cells in vitro) were assessed. Human peripheral blood mononuclearcells (PBMCs) were isolated from blood filters. After separation by celldensity gradient, mononuclear cells were collected and washed twice withHank's balanced salt solution. Cells were then resuspended in Iscove'sModified Dulbecco's Medium (IMDM, Sigma) supplemented with 10% foetalcalf serum (Bioconcept, Paris, france), 1% L-glutamine (Sigma), 1%penicillin/streptomycin (Sigma) and 0.1% gentamycin (Sigma). PBMCs(7×10⁵ cells/well) were then incubated with live and heat treatedbacteria (equivalent 7×10⁶ cfu/well) in 48 well plates for 36 h. Theeffects of live and heat treated bacteria were tested on PBMCs from 8individual donors splitted into two separate experiments. After 36 hincubation, culture plates were frozen and kept at −20° C. untilcytokine measurement. Cytokine profiling was performed in parallel (i.e.in the same experiment on the same batch of PBMCs) for live bacteria andtheir heat-treated counterparts.

Levels of cytokines (IFN-γ, IL-12p40, TNF-α and IL-10) in cell culturesupernatants after 36 h incubation were determined by ELISA (R&D DuoSetHuman IL-10, BD OptEIA Human IL12p40, BD OptEIA Human TNF, BD OptEIAHuman IFN-γ) following manufacturer's instructions. IFN-γ, IL-12p40 andTNF-α are pro-inflammatory cytokines, whereas IL-10 is a potentanti-inflammatory mediator. Results are expressed as means (pg/ml)+/−SEMof 4 individual donors and are representative of two individualexperiments performed with 4 donors each.

In Vivo Effect of Live and Heat Treated Bifidobacterium breve NCC2950 inPrevention of Allergic Diarrhea

A mouse model of allergic diarrhea was used to test the Th1 promotingeffect of B. breve NCC2950 (Brandt E. B et al. JCI 2003; 112(11):1666-1667). Following sensitization (2 intraperitoneal injections ofOvalbumin (OVA) and aluminium potassium sulphate at an interval of 14days; days 0 and 14) male Balb/c mice were orally challenged with OVAfor 6 times (days 27, 29, 32, 34, 36, 39) resulting in transientclinical symptoms (diarrhea) and changes of immune parameters (plasmaconcentration of total IgE, OVA specific IgE, mouse mast cell protease1, i.e MMCP-1). Bifidobacterium breve NCC2950 live or heat treated at90° C. for 30 min, was administered by gavage 4 days prior to OVAsensitization (days −3, −2, −1, 0 and days 11, 12, 13 and 14) and duringthe challenge period (days 23 to 39). A daily bacterial dose of around10⁹ colony forming units (cfu) or equivalent cfu/mouse was used.

Results

Induction of Secretion of ‘Pro-Inflammatory’ Cytokines After HeatTreatment

The ability of heat treated bacterial strains to stimulate cytokinesecretion by human peripheral blood mononuclear cells (PBMCs) wasassessed in vitro. The immune profiles based on four cytokines uponstimulation of PBMCs by heat treated bacteria were compared to thatinduced by live bacterial cells in the same in vitro assay.

The heat treated preparations were plated and assessed for the absenceof any viable counts. Heat treated bacterial preparations did notproduce colonies after plating.

Live probiotics induced different and strain dependent levels ofcytokine production when incubated with human PBMCs (FIG. 8). Heattreatment of probiotics modified the levels of cytokines produced byPBMCs as compared to their live counterparts. Heat treated bacteriainduced more pro-inflammatory cytokines (TNF-α, IFN-γ, IL-12p40) thantheir live counterparts do. By contrast heat treated bacteria inducedsimilar or lower amounts of IL-10 compared to live cells (FIG. 8). Thesedata show that heat treated bacteria are more able to stimulate theimmune system than their live counterparts and therefore are more ableto boost weakened immune defences. In other words the in vitro dataillustrate an enhanced immune boost effect of bacterial strains afterheat treatment.

In order to illustrate the enhanced effect of heat-treated B. breveNCC2950 (compared to live cells) on the immune system, both live andheat treated B. breve NCC2950 (strain A) were tested in an animal modelof allergic diarrhea.

As compared to the positive control group, the intensity of diarrhea wassignificantly and consistently decreased after treatment with heattreated B. breve NCC2950 (41.1%±4.8) whereas the intensity of diarrheawas lowered by only 20±28.3% after treatment with live B. breve NCC2950.These results demonstrate that heat-treated B. breve NCC2950 exhibits anenhanced protective effect against allergic diarrhea than its livecounterpart (FIG. 9).

As a consequence, the ability of probiotics to enhance the immunedefenses was shown to be improved after heat treatment.

EXAMPLES 3-6

The following infant feeding formulas may be prepared:

Protein 1.83 1.83 2.699 2.25 (g/100 kcal) Whey/Casein 70/30 70/30 23/7745/55 CHO 11.2 11.2 10.2 11.3 (g/100 kcal) Lactose 11.16 11.2 7.11 4.5(g/100 kcal) Maltodextrine — — 3.11 2.9 (g/100 kcal) Fat 5.34 5.3 5.375.1 (g/100 kcal) Probiotics/g 10⁹ cfu 10⁹ cfu heat 10⁹ cfu UHT 10⁹ cfuUHT dry weight Lactobacillus treated treated treated johnsonii (75° C.,20 min) Lactobacillus Bifidobacterium La1 Bifidobacterium johnsoniibreve longum La1 NCC 2950 NCC 3001 LC-PUFA ARA/DHA ARA/DHA — — (0.23g/100 g FA) Nucleotides 2 2 — — mg/100 mL Energy 65 65 64.84 65 Kcal/100mL

The invention claimed is:
 1. An infant feeding formula to beadministered to infants as an only nutrition source or as acomplementary nutrition source in addition to breastfeeding, to providecomplete nutrition to the infant and also comprising non-replicatingprobiotic micro-organisms rendered non-replicating by a heat treatmentat 120 to 140° C. for 5-15 seconds, the non-replicating probioticmicro-organisms selected from the group consisting of Bifidobacteriumlongum NCC 2705, Bifidobacterium breve NCC 2950, Lactobacillus johnsoniiLa1, Lactobacillus reuteri DSM17938, Lactobacillus reuteri ATCC55730,Streptococcus thermophilus NCC 2019, Streptococcus thermophilus NCC2059, Lactobacillus casei NCC 4006, Lactobacillus acidophilus NCC 3009,Lactobacillus casei ACA-DC 6002 (NCC 1825), Escherichia coli Nissle,Lactobacillus bulgaricus NCC 15, Lactococcus lactis NCC 2287, andcombinations thereof, wherein the non-replicating probioticmicro-organisms induce less pro-inflammatory cytokines while maintainingor inducing additional Interleukin 10 (IL-10) production relative tolive counterpart.
 2. The infant feeding formula in accordance with claim1 having a caloric density of 62-68 kcal/100 ml, and comprising aprotein source in an amount of 1.5-2.8 g/100 kcal, a carbohydrate sourcein an amount of 10-12 g/100 kcal, and a lipid source in an amount of5-5.5 g/100 kcal.
 3. The infant feeding formula in accordance with claim1, comprising 0.2-0.3 g LC-PUFA/100 g fatty acids, wherein the LC-PUFAare a combination of ARA and DHA.
 4. The infant feeding formula inaccordance with claim 1, comprising 1.5-2.5 mg nucleotides per 100 mLformula.
 5. The infant feeding formula in accordance with claim 1,comprising the non-replicating probiotic micro-organisms in an amountcorresponding to 10⁴ to 10¹² cfu.
 6. A method for the treatment ofinflammatory disorders, the method comprising: administering to aninfant the infant feeding formula of claim
 1. 7. A method for thetreatment of disorders related to a compromised immune defense, themethod comprising: administering to an infant the infant feeding formulaof claim
 1. 8. The infant feeding formula in accordance with claim 1further comprising a live counterpart of the non-replicating probioticmicro-organisms.
 9. The infant feeding formula in accordance with claim1, containing about 0.005 mg-1000 mg of the non: replicatingmicro-organisms per daily dose.
 10. The method in accordance with claim6, wherein the infant formula has a caloric density in the range of62-68 kcal/100 ml and further comprises a protein source in an amount of1.5-2.8 g/100 kcal, a carbohydrate source in an amount of 10-12 g/100kcal, and a lipid source in an amount of 5-5.5 g/100 kcal.
 11. Themethod in accordance with claim 6, wherein the infant formula comprisesabout 0.005 mg-1000 mg non-replicating micro-organisms per daily dose.12. The method in accordance with claim 7, wherein the infant formulahas a caloric density in the range of 62-68 kcal/100 ml and furthercomprises a protein source in an amount of 1.5-2.8 g/100 kcal, acarbohydrate source in an amount of 10-12 g/100 kcal, and a lipid sourcein an amount of 5-5.5 g/100 kcal.
 13. The method in accordance withclaim 7, wherein the infant formula comprises about 0.005 mg-1000 mgnon-replicating micro-organisms per daily dose.